Summary Primary ovarian insufficiency (POI) is part of the continuum of ovarian dysfunction ranging from infertility with a high FSH level to early menopause before age 45 years, and affects 5-10% of women. Using next generation sequencing in women with familial POI, we have identified novel, heterozygous, stop gained mutations in two families. The first was in the eukaryotic translation initiation factor transport protein eIF4ENIF1, which causes menopause 20 years earlier than the population average. The second was in POLR2C, the third largest subunit of RNA polymerase II, which appears to cause earlier menopause in each generation. We will now apply unique DNA sequence analysis software developed at the University of Utah to discover novel gene mutations in sporadic POI and familial POI cases. In addition, we will examine the familial segregation of POI and comorbid disease. Finally, we will use a mouse model of the eIF4ENIF1 stop gained mutation to identify genes important for oocyte growth and meiosis. Specific Aim 1 will determine the breadth of genetic mutations in large populations of women with POI that have undergone whole exome sequencing. We will use novel software (VAAST, pVAAST and Phevor) developed at the University of Utah and controls recruited for health in old age to prioritize variants in two POI cohorts with replication in a third (n~300). The software identifies rare, damaging variants in genes that have a strong relationship to the POI phenotype. Specific Aim 2 will identify familial cases of POI in the Utah Population Database, the most extensive genealogical database in the U.S. The inheritance pattern, associated phenotypes and comorbid diseases will be analyzed in familial POI to determine its effect on overall health and to determine families at risk for POI. Specific Aim 3 will determine the mechanism of ovarian insufficiency in carriers of the eIF4ENIF1 stop gained mutation using a mouse model. The differentially translated mRNA in polysomes in the presence of the stop gained mutation compared to wild type will be determined. The differentially translated mRNAs in the eIF4ENIF1 stop gained mouse model will highlight the genes important for oocyte development and meiosis, and will inform the search for gene mutations in Specific Aim 1. The work will address two research gaps by illuminating our understanding of the genetics of the reproductive aging transition. It will also address fertility as a marker of overall health by identifying diseases associated with decreased ovarian reserve and POI. We will then be able to use family history and associated disorders to identify women at risk for POI. New gene mutations and pathways will inform software algorithms such as Phevor, which will use the new information to prioritize variants discovered in next generation sequencing to determine the genetic cause of POI in additional women. Early identification will bring the potential to preserve fertility and create targeted treatment options for these women.